Cell penetrating peptides for intracellular delivery of molecules

Abstract

A cell-penetrating peptide characterized in that it comprises an amino acid sequence X 3 X 4 X 1 X 2 X 5 X 4 X 1 X 2 X 6 X 7 X 1 X 8 X 9 X 10 X 11 X 12 X 13 (SEQ ID No: 11), wherein X 1 is F or W, X 2 is F, W or Y, X 3 is beta-A or S, X 4 is K, R or L, X 5 is E, R or S, X 6 is R. T or S, X 7 is E, R or S, X 8 is none, F or W, X 9 is P or R, X 10 is R or L, X 11 is K, W or R, X 12 is R or F and X 13 is R or K.

The invention claimed is: 
     
       1. A cell-penetrating peptide characterized in that it comprises an amino acid sequence X 3 X 4 X 1 X 2 X 5 X 4 X 1 X 2 X 6 X 7 X 1 X 8 X 9 X 10 X 11 X 12 X 13  (SEQ ID No: 11), wherein X 1  in positions 3, 7, and 11 are, independently from each other, F or W, X 2  in positions 4 and 8 are, independently from each other, F, W or Y, X 3  is beta-A or S, X 4  in positions 2 and 6 are, independently from each other, K, R or L, X 5  is E, R or S, X 6  is R, T or S, X 7  is E, R or S, X 8  is none, F or W, X 9  is P or R, X 10  is R or L, X 11  is K, W or R, X 12  is R or F and X 13  is R or K. 
     
     
       2. The cell-penetrating peptide of  claim 1 , characterized in that it comprises an amino acid sequence X 3 X 4 WX 2 EX 4 WX 2 X 6 X 7 X 1 PRX 11 RX 13  (SEQ ID No: 12), wherein X 1  is F or W, X 2  in positions 4 and 8 are, independently from each other, F, W or Y, X 3  is beta-A or S, X 4  in positions 2 and 6 are, independently from each other, K or R, X 6  is T or R, X 7  is E or R, X 11  is R or K, and X 13  is R or K. 
     
     
       3. The cell-penetrating peptide of  claim 1 , wherein the amino acid sequence is selected from the group consisting of: 
       
         
           
                 
               
                   (SEQ ID No: 1) 
                 
                 
                 
               
                     
                   X 3 KWFERWFREWPRKRR, 
                 
                     
                 
                 
               
                   (SEQ ID No: 2) 
                 
                 
                 
               
                     
                   X 3 KWWERWWREWPRKRK, 
                 
                     
                 
                 
               
                   (SEQ ID No: 3) 
                 
                 
                 
               
                     
                   X 3 RWWEKWWTRWPRKRK, 
                 
                     
                   and 
                 
                     
                 
                 
               
                   (SEQ ID No: 4) 
                 
                 
                 
               
                     
                   X 3 RWYEKWYTEFPRRRR, 
                 
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
                
               
            
             
                
               
            
             
                
               
            
           
         
         wherein X 3  is beta-A or S. 
       
     
     
       4. A cell-penetrating peptide characterized in that it comprises the amino acid sequence X 3 KX 14 WWERWWRX 14 WPRKRK (SEQ ID No: 9), wherein X 3  is a beta-alanine or a serine, X 14  in positions 3 and 11 are, independently from each other, a non-natural amino acid, and there is a hydrocarbon linkage between the two non-natural amino acids. 
     
     
       5. The cell-penetrating peptide of  claim 1 , characterized in that it comprises an amino acid sequence: X 3 X 4 X 1 WX 5 X 4 X 1 WX 6 X 7 WX 8 X 9 X 10 WX 12 R (SEQ ID No: 13), wherein X 1  in positions 3 and 7 are, independently from each other, F or W, X 3  is beta-A or S, X 4  in position 2 is K, R or L, X 4  in position 6 is L or R, X 5  is R or S, X 6  is R or S, X 7  is R or S, X 8  is F or W, X 9  is R or P, X 10  is L or R, and X 12  is R or F. 
     
     
       6. The cell-penetrating peptide of  claim 1 , wherein the amino acid sequence is selected from the group consisting of: 
       
         
           
                 
               
                   (SEQ ID No: 5) 
                 
                 
                 
               
                     
                   X 3 RWWRLWWRSWFRLWRR, 
                 
                     
                 
                 
               
                   (SEQ ID No: 6) 
                 
                 
                 
               
                     
                   X 3 LWWRRWWSRWWPRWRR, 
                 
                     
                 
                 
               
                   (SEQ ID No: 7) 
                 
                 
                 
               
                     
                   X 3 LWWSRWWRSWFRLWFR, 
                 
                     
                   and 
                 
                     
                 
                 
               
                   (SEQ ID No: 8) 
                 
                 
                 
               
                     
                   X 3 KFWSRFWRSWFRLWRR, 
                 
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
                
               
            
             
                
               
            
             
                
               
            
           
         
         wherein X 3  is beta-A or S. 
       
     
     
       7. A cell-penetrating peptide derived from the amino acid sequence X 3 X 4 X 1 WX 5 X 4 X 1 WX 6 X 7 WX 8 X 9 X 10 WX 12 R (SEQ ID No: 13) by replacement of the amino acids in positions 5 and 12 of SEQ ID No: 13 by non-natural amino acids, wherein X 1  in positions 3 and 7 are, independently from each other, F or W, X 3  is beta-A or S, X 4  in position 2 is K, R or L, X 4  in position 6 is L or R, X 5  is R or S, X 6  is R or S, X 7  is R or S, X 8  is F or W, X 9  is R or P, X 10  is L or R, and X 12  is R or F, and wherein there is a hydrocarbon linkage between the two non-natural amino acids. 
     
     
       8. The cell-penetrating peptide of  claim 7 , characterized in that it comprises the amino acids sequence X 3 RWWX 14 LWWRSWX 14 RLWRR (SEQ ID No: 10), wherein X 3  is a beta-alanine or a serine, X 14  in positions 5 and 12 are, independently from each other, a non-natural amino acid, and there is a hydrocarbon linkage between the two non-natural amino acids. 
     
     
       9. The cell-penetrating peptide of  claim 1 , further comprising, covalently linked to the N-terminal end of the amino acid sequence, one or several chemical entities selected from the group consisting of an acetyl, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide and a targeting molecule. 
     
     
       10. The cell-penetrating peptide of  claim 1 , further comprising, covalently linked to the C-terminal end of said amino acid sequence, one or several groups selected from the group consisting of a cysteamide, a cysteine, a thiol, an amide, an optionally substituted nitrilotriacetic acid, a carboxyl, a linear or branched optionally substituted C 1 -C 6  alkyl, a primary or secondary amine, a lipid, a phospholipid, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide and a targeting molecule. 
     
     
       11. A complex comprising a cell-penetrating peptide according to  claim 1  and a cargo selected from the group consisting of peptides, proteins, peptide analogs, uncharged oligonucleotides, PNAs and small hydrophobic molecules. 
     
     
       12. The complex of  claim 11 , wherein said cargo is a molecule of at most 1.5 kDa. 
     
     
       13. The complex of  claim 11 , wherein said cargo is an anticancer or an anti-viral drug. 
     
     
       14. The complex of  claim 11 , wherein said cargo is selected from the group consisting of amino acids, di- or tri-peptides, daunomycin, Paclitaxel, doxorubicin, AZT, porphyrin, fluorescently labelled nucleosides or nucleotides, hydrophobic maghemite and fluorescent dyes. 
     
     
       15. The complex of  claim 11 , wherein the size of the complex is between 50 and 300 nm. 
     
     
       16. A nanoparticle comprising a complex according to  claim 11 , coated by a layer of peripheral cell-penetrating peptides, wherein said peripheral cell-penetrating peptides have a peptide sequence different from SEQ ID No: 11. 
     
     
       17. A nanoparticle comprising a core which comprises a cargo complexed to a first entity selected from the group consisting of cell-penetrating peptides, liposomes, polycationic structures and carbon nanoparticles, wherein said core is coated by peripheral cell-penetrating peptides, and wherein the peripheral cell-penetrating peptides have the peptide sequence of a cell-penetrating peptide according to  claim 1 . 
     
     
       18. The nanoparticle of  claim 17 , wherein said first entity is a cell-penetrating peptide selected from the group consisting of VEPEP-6a (SEQ ID No: 29), VEPEP-6b (SEQ ID No: 30), VEPEP-6c (SEQ ID No: 31), VEPEP-6d (SEQ ID No: 32), VEPEP-6e (SEQ ID No: 33), VEPEP-6f (SEQ ID No: 34), VEPEP-9a1 (SEQ ID No: 35), VEPEP-9a2 (SEQ ID No: 36), VEPEP-9b1 (SEQ ID No: 37), VEPEP-9b2 (SEQ ID No: 38), VEPEP-9c1 (SEQ ID No: 39), VEPEP-9c2 (SEQ ID No: 40), CADY, MPG, PEP-1, PPTG 1, and poly Arginine. 
     
     
       19. The nanoparticle of  claim 17 , wherein the size of the nanoparticle is between 20 and 300 nm. 
     
     
       20. The nanoparticle of  claim 17 , wherein the peripheral cell-penetrating peptides comprise a poly-ethylene glycol group covalently linked to their N-terminus, and/or a cysteamide group covalently linked to their C-terminus. 
     
     
       21. The nanoparticle of  claim 17 , wherein at least some of the peripheral cell-penetrating peptides are bound to a targeting molecule. 
     
     
       22. The nanoparticle of  claim 17  for use as a medicament. 
     
     
       23. The nanoparticle of  claim 17 , for use as a marker or an imaging agent. 
     
     
       24. The nanoparticle of  claim 17 , for use in the treatment of a brain disease. 
     
     
       25. The nanoparticle of  claim 17  for use in the treatment of a lymph node disease. 
     
     
       26. A therapeutic, cosmetic or diagnostic composition comprising a nanoparticle according to  claim 17 . 
     
     
       27. The composition of  claim 26 , which is formulated for intravenous, intratumoral, topical, intrarectal, intranasal, transdermal, or intradermal administration, or for administration via a mouth spray, or for administration as a subcutaneous implant for slow release of a drug. 
     
     
       28. A method for delivering a molecule into a cell in vitro, comprising a step of putting said cell into contact with a nanoparticle according to  claim 17 , wherein the cargo of the nanoparticle is said molecule.